Program Director/Principal Investigator (Last, First, Middle): Ip, Blanche C. PROJECT SUMMARY: Ideally, patients with tissue and organ failure would be treated using biologics of human origin, but there exists an acute shortage of transplantable organs and available tissue repair materials. Injectable biologically complex hydrogels, including extracellular matrix (ECM) formulated from extracts of healthy tissues, are emerging as a promising new treatment. ECM can be extract from non-human tissues, But, the animals ECM is composed of proteins foreign to humans that limits their effectiveness or even causes further tissue damage. Healthy human hearts are not a source of ECM because they are too valuable for transplantation and cadaver hearts are unsuitable. An intriguing alternative could be ECM secreted by human cells cultured in vitro. However, the biochemical composition and physical properties of cell-secreted ECM are highly sensitive to culture conditions, of which this basic science is poorly understood. Therefore, cells cultured on scaffolds (of natural or synthetic polymers) or non-physiological stiff plastic surfaces with reduced cell densities, decreased cell-cell contact and altered cell-matrix adhesions may generate ECM with differing composition and therapeutic efficacy. Needed is a new scaffold-free method of generating human ECM to support tissue repair and regeneration. Our long-term goal is to develop therapeutic human ECM from lab-grown three-dimensional (3D) human tissues from human cell lines that are biofabricated without the use of scaffolds or non-physiological stiff surfaces. Our central hypothesis is that the human cardiac ECM from our lab-grown 3D scaffold-free tissues will exhibit overall composition and mechanics more consistent with ECM from native human cardiac tissue relative to ECM from porcine cardiac tissue and 3D cardiac cell-sheets. PI: Ip and Collaborator: Morgan have preliminary data and extensive complementary expertise for the proposed study to accomplish study goals which require 1) a robust technological platform that can generate stable 3D scaffold-free cardiac tissue, where cells do not interact with polymeric scaffolds or tissue culture plastic, 2) an optimized decellularization protocol to generate decellularized ECM that is free of cells and DNA and retains the native protein composition and structure, 3) thorough evaluation in the quality and injectability of ECM generated from our 3D scaffold-free tissue and contrast the results with ECM from 3D cell sheet grown on stiff plastic surface, and from native human and porcine cardiac tissue. In this R03, we will utilize our patented micro-mold technology to generate stable 3D scaffold-free human cardiac tissue with human cardiac fibroblasts, cardiomyocytes and cardiac microvascular endothelial cells and evaluate three decellularized protocols to generate optimal quality decellularized ECM in Aim 1. We will then develop, optimize and execute three complementary methods to evaluate protein composition between the different ECM, as well as to examine the quantity and injectability of the ECM in Aim 2. We envision these technologies can have other bioengineering applications to enhance human biomimicry, including microfluidic devices to study disease progression such as tumor invasion.